Circular smooth muscle from the body of the esophagus is normally relaxed and contracts in response to neural input. In the cat esophagus, which closely mimics the human one, neurons releasing acetylcholine (ACh) initiate contraction and neurons secreting vasoactive intestinal peptide (VIP) inhibit contraction. Contraction in response to ACh requires Ca++ influx from the extracellular solution. Application of ACh to freshly isolated cells does not elicit action potentials or even a depolarization but rather a transient hyperpolarization (followed by a small and gradual depolarization). In our pilot studies, we have seen no evidence of inward Ca++ or cationic currents in response to cholinergic stimulation. How ACh causes an elevation in intracellular Ca++ in these cells is unknown and is a central issue in this proposed investigation. In addition, until our pilot studies we had no reason to suspect that these cells had any viable intracellular Ca++ stores at all. Inositol 1,4,5- trisphosphate has no effect and is not generated in response to ACh. The role of these stores, their relationship to the required Ca++ influx, their possible dependence on extracellular Ca++, and the agent(s) responsible for triggering the observed release of Ca++ are all unknown and will be investigated. Using patch clamp techniques and simultaneously high time resolution measurement of intracellular Ca++ with Fura-2 we will: 1. Examine the dependence of intracellular storage sites on extracellular Ca++. 2. Determine the contributions to the total rise in cytosolic Ca++ made by intracellular and extracellular sources in response to ACh. We will investigate the role of the transient hyperpolarizations in causing Ca++ influx through voltage activated Ca++ channels 3. Determine how Ca++ is released from stores in response to ACh. 4. Identify the mechanisms and agents involved in causing the transient hyperpolarizations elicited by ACh. VIP blocks contraction in response to ACh. How it does this is unknown. Our pilot studies suggest that VIP inhibits voltage activated Ca++ channels by a mechanism which involves the elevation of intracellular adenosine 3':5' cyclic monophosphate (cyclic AMP). In other vascular and visceral smooth muscle preparations, investigators have shown that elevations in intracellular cyclic nucleotides decrease resting and agonist stimulated Ca++ levels. We will investigate the mechanisms by which VIP and cyclic nucleotides control cytosolic Ca++. Specifically we will: 1. Determine how VIP inhibits voltage activated Ca++ channels. 2. Investigate the effects of VIP and cyclic nucleotides on release and uptake of Ca++ into intracellular storage sites. 3. Investigate the effects of VIP and cyclic nucleotides on the membrane potential and the transient hyperpolarizations which occur in response to ACh. This investigation will provide substantial new information about the mechanisms by which neurotransmitters and intracellular second messengers regulate ion channels, intracellular Ca++, and contraction in esophageal circular smooth muscle.